The present invention relates to a method for producing breads through use of yeast that has been transformed with a photoprotein expression recombinant vector.
At present, a variety of yeast strains are used in the bread industry, in consideration of consistent activity of yeast during continuous bread making steps, as well as staling resistance and uniform quality of the resultant breads. Recently, a number of methods have been proposed in an attempt to produce breads having a soft flavor of fermentation and being endowed with excellent internal grain and external appearance.
However, no method has ever attained these targets successfully.
In view of the foregoing, the present inventors have performed careful studies so as to establish a method for producing breads having a soft flavor of fermentation and exhibiting excellent internal structure and external appearance, thereby leading to completion of the present invention.
Accordingly, the present invention provides a method for producing breads by use of a yeast which has been transformed with a photoprotein expression recombinant vector, the vector containing a fragment of a yeast-derived agglutinin gene and a gene coding for a photoprotein.
The present invention will next be described in detail.
Examples of the predominant bread-making material for producing the breads of the present invention include strong flour, semi-strong flour, medium flour, weak flour, durum wheat flour, durum semolina, rye flour, oat flour, barley flour, and mixtures thereof.
The present invention employs a specific type of yeast; i.e., a yeast which has been transformed with a photoprotein expression recombinant vector containing a fragment of a yeast-derived agglutinin gene and a gene coding for a photoprotein (the yeast is hereafter referred to as the EGFP yeast).
The above-mentioned fragment of an agglutinin gene may be a DNA fragment containing a nucleotide sequence coding for 320 amino acid residues starting from the C-terminal of xcex1-agglutinin derived from a microorganism which belongs to genus Saccharomyces and a 3xe2x80x2-nontranslational region composed of 446 bases. This DNA fragment also contains a signal region for GPI anchoring. This DNA fragment may be obtained by cleaving, with a restriction endonuclease, a plasmid pGA11 carrying the mentioned DNA fragment, the plasmid being disclosed by Murai, T. et al., in Applied and Environmental Microbiology, 63, 1362-1366 (1997).
No particular limitation is imposed on the photoprotein, so long as it issues luminescence or fluorescence. Examples of the photoprotein include luciferase, aequorin, and green fluorescent protein (GFP). In particular, green fluorescent protein derived from Aequorea Victoria is preferred.
The photoprotein expression vector can be obtained through inserting the aforementioned gene into an expression vector which is usually used in processes making use of yeast. Examples of preferred expression vectors include, but are not limited to, pYE22M (Sawani-Hatanaka, H., et al., Biosci. Biotechnol. Biochem., 59, 1221-1228 (1995)) and pRS404 (Robert, S., S. and Philip Hieter, Genetics, 122, 19-27 (1989)).
The above-described photoprotein expression recombinant vector preferably contains a secretion signal region, a structural gene of the photoprotein (e.g., green fluorescent protein), and a fragmentary region of the yeast-derived agglutinin gene (e.g., a DNA fragment containing a nucleotide sequence coding for 320 amino acid residues starting from the C-terminal of xcex1-agglutinin and a 3xe2x80x2-nontranslational region composed of 446 bases), in this order of arrangement.
An example EGFP yeast is Saccharomyces cerevisiae MT8-1 (pICS:GFP). Detailed description of EGFP yeast strains and a method for their creation is described in, among other references, Japanese Patent Application Laid-Open (kokai) No. 2000-102387.
The EGFP yeast strains may be used singly or in combination with any other yeast strain.
The amount of the EGFP yeast falls within a range of 0.01 to 10% by weight on the basis of the amount of farina, preferably 0.5 to 8% by weight, more preferably 2 to 5% by weight.
Other auxiliary raw materials include yeast food, sugars, common salt, oils or fats, egg, and dairy products, which are appropriately selected according to needs.
Examples of sugars include uncured sugar, cured sugar, liquid sugar, fructose, invert sugar, and starch sugar.
Examples of oils or fats include butter, margarine, shortening, and lard.
Examples of egg include frozen eggs, stirred egg, dried egg, and concentrated egg.
Examples of dairy products include fresh milk, powdery milk, condensed milk, cheese, and fresh cream.
In the present invention, any conventional bread-making process may be employed conveniently. For example, the following processes are employable: a rapid fermentation method, a straight dough method, a sponge dough method, a pre-ferment method, a sour dough method, a saketane leaven method, a hop leaven method, a Chumen process, a Chorleywood process, a continuous bread making process, and a retarded dough method.
The frozen dough method may be further classified into a plate-dough freezing method, in which a dough is frozen immediately after kneading; a round-dough freezing method, in which a divided and rounded dough is frozen; a molding-freezing method, in which a dough is frozen after it has undergone molding; and a proven-dough freezing method, in which a dough that has undergone final proofing is frozen. Any of these methods may be employed in the present invention.
The breads obtained by the method of the present invention include pullman-type bread, pastries, Danish pastries, French breads, rye breads, croissants, butter rolls, sweet rolls, brioches, yeast doughnuts, pizza pies, and manju (ingredient-containing buns).